Encoding of contextual fear memory in hippocampal–amygdala circuit

ARTICLE https://doi.org/10.1038/s41467-020-15121-2 OPEN Encoding of contextual fear memory in hippocampal–amygdala circuit 1234567890():,; Woong Bin Kim1 & Jun-Hyeong Cho 1✉ In contextual fear conditioning, experimental subjects learn to associate a neutral context with an aversive stimulus and display fear responses to a context that predicts danger. Although the hippocampal–amygdala pathway has been implicated in the retrieval of contextual fear memory, the mechanism by which fear memory is encoded in this circuit has not been investigated. Here, we show that activity in the ventral CA1 (vCA1) hippocampal projections to the basal amygdala (BA), paired with aversive stimuli, contributes to encoding conditioned fear memory. Contextual fear conditioning induced selective strengthening of a subset of vCA1–BA synapses, which was prevented under anisomycin-induced retrograde amnesia. Moreover, a subpopulation of BA neurons receives stronger monosynaptic inputs from context-responding vCA1 neurons, whose activity was required for contextual fear learning and synaptic potentiation in the vCA1–BA pathway. Our study suggests that synaptic strengthening of vCA1 inputs conveying contextual information to a subset of BA neurons contributes to encoding adaptive fear memory for the threat-predictive context. 1 Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA 92521, USA. ✉email: NATURE COMMUNICATIONS | (2020)11:1382 | https://doi.org/10.1038/s41467-020-15121-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15121-2 I n order to survive, animals develop fear responses to dangerous situations. The neural mechanism of learned fear has great survival value for animals, which must predict danger from seemingly neutral contexts. In contextual fear conditioning, an experimental model of fear learning, experimental subjects learn to associate a neutral context with an aversive stimulus and display fear responses to a context that predicts danger1. Contextual fear learning requires coordinated activity of the hippocampus and amygdala2. Ventral CA1 (vCA1) hippocampal neurons encode and convey contextual representations through monosynaptic projections to the amygdala, which induces defensive behavior3–5. Thus, the vCA1–amygdala pathway can play an essential role in contextual fear learning. Although the vCA1–amygdala pathway has been implicated in the retrieval of contextual fear memory4, the mechanism by which contextual fear memory is encoded in this circuit has not been investigated. Exposure to a context activates a subset of vCA1 hippocampal neurons, which convey contextual representations directly to the amygdala3. The contextual information is then integrated with aversive signals in the amygdala for fear memory formation1,2. Strengthening of the hippocampal–amygdala pathway as a consequence of learning can facilitate the activation of the amygdala, resulting in conditioned fear responses to the threat-predictive context during the recall of contextual fear memory6. Moreover, selective strengthening of the hippocampal inputs conveying specific contextual information to the amygdala can confer selective fear responses only to the relevant context7. However, these hypotheses have not been examined in contextual fear conditioning. Recent studies have identified memory engram cells in the hippocampus and amygdala8–13. Although these studies demonstrate the role of memory engram cells in contextual fear learning, it remains unknown how memory engram cells in the amygdala are connected to hippocampal engram cells encoding specific contextual representations, as well as how the synaptic strength of these connections is modified to encode contextual fear memory. In this study, we determined the mechanism by which contextual fear memory is encoded in the hippocampal–amygdala circuit by testing our hypothesis that fear memory associated with a particular context is encoded by selective strengthening of hippocampal inputs conveying the contextual information to the amygdala. Results vCA1–BA activity contributes to contextual fear learning. In the anterograde tracing experiment, eYFP-labeled vCA1 projections were found in the basolateral (BLA) and basomedial nuclei of the amygdala (BMA), collectively termed the basal amygdala (BA) (Fig. 1a, b). In the retrograde tracing experiment, hippocampal neurons projecting to the BA were predominantly found in the vCA1 and ventral subiculum (Fig. 1c, d), suggesting monosynaptic connection of vCA1 neurons to BA neurons3. More vCA1 neurons projecting to the BA (vCA1:BA projectors) expressed the immediate early gene c-fos in mice that were exposed to a novel context or recalled contextual fear memory than in mice left in their home cages (Fig. 1c, d, Supplementary Fig. 1a, and Supplementary Table 1), suggesting that a subset of vCA1: BA projectors can encode contextual representations. We next determined the role of the vCA1–BA pathway in the formation of contextual fear memory using a chemogenetic approach (Fig. 1e, f). Application of clozapine N-oxide (CNO) induced hyperpolarization and inhibited action potential (AP) firing in vCA1: BA projectors expressing hM4Di (Supplementary Fig. 2a–c), indicating the validity of our approach to silence vCA1–BA activity. After surgery, mice received a CNO injection and received unconditioned stimuli (US) in Context A 30 min 2 later (Fig. 1g and Supplementary Fig. 1a). After 24 h, mice were tested for freezing behavior in Context A. On the following day, mice were fear conditioned in Context A after a vehicle injection and tested for freezing behavior in Context A 24 h later. In the hM4Di group, mice displayed significantly reduced freezing behavior when they had received a CNO injection on the training day compared with a vehicle injection, whereas in the mCherry group, there was no difference in freezing behavior on the test days between CNO and vehicle injections on the training day (Fig. 1h). The CNO effect in the hM4Di group on conditioned fear response was not due to the order of CNO and vehicle injections before fear conditioning (Supplementary Fig. 2d–e). These results indicate that silencing vCA1–BA activity during contextual fear learning decreased conditioned fear responses to the context 24 h later. Thus, vCA1–BA activity contributes to the acquisition of contextual fear memory14. vCA1–BA activity paired with shocks generated a fear memory. We next examined whether the activation of a random population of vCA1: BA projectors could serve as a conditioned stimulus with which mice could learn to associate an aversive stimulus. We induced Chronos or eYFP expression in vCA1: BA projectors and implanted an optical cannula to the vCA1 for in vivo photostimulation (Fig. 2a, b). Blue light illumination at 20 Hz reliably induced AP firing in Chronos-expressing vCA1 neurons (Supplementary Fig. 3). After surgery, (...truncated)